Introduction: Smooth muscle cells (SMC) are valuable cell sources for fabricating a functional tissue engineered blood vessel (TEBV) as these cells play a major role in regulation of vessel tone and blood pressure, as well as maintenance of a functional endothelium. Unfortunately human SMC are a difficult cell source to access and are not easily expandable as they undergo senescence within a few passages following their initial isolation. Direct transdifferentiation, the process of converting a somatic cell into another type of somatic cell without going through an intermediate pluripotent stage, is a robust alternative for obtaining SMC source. In this study, we have successfully transdifferentiated human endothelial progenitors (EPC) into functional induced SMC (iSMC) by overexpressing myocardin (MYOCD) gene. Finally, the utility of these iSMC in the assembly of TEBV is demonstrated.
Materials and Methods: EPC were isolated from the umbilical cord blood obtained from the Carolina Cord Blood Bank as described by previous studies[1]. DNA vector and lentiviral particles for the lentiviral delivery of MYOCD into EPCs were prepared as previously described[2]. We used immunofluorescence, flow cytometry, and microarray gene expression analysis to assess the phenotypic reorganization of iSMC as compared to the EPC controls. We also measured the calcium signaling activity using R-GECO to evaluate their functional phenotypic characteristics. Lastly, we have embedded iSMC in a dense collagen gel to fabricate a tubular vessel construct. The vasoactivity of the TEBV in response to flow rate change and drug administration was measured to assess iSMC function in physiologically relevant environment.
Results: Upon lentiviral transduction, EPC rapidly transdifferentiated into iSMC within 7 days post transduction. Phase contrast image of iSMC showed a transformation of cells into elongated and spindle-like morphology often seen in SMC. Immunofluorescent staining and quantitative RT-PCR analysis showed that SMC related genes, such as MYH11, were uregulated by an average of 8-fold and endothelial cell related genes, such as CD31, were downregulated by an average of 2-fold in iSMC as compared to the control EPC. In addition, the whole genome microarray analysis demonstrated that genes associated with pathways controlling smooth muscle differentiation were significantly upregulated in iSMC compared to EPC (p<0.05), and were closer to native SMC expression. The calcium signaling activity displayed that the addition of 100µM phenylephrine, a vasoconstrictor drug, induced a steep transient increase of 75.6% in the fluorescence intensity in iSMC at 2 weeks post-MYOCD induction (Figure 1), but not in the control EPCs. Finally, TEBV containing iSMC constricted by 2.0 ± 0.25% in response to 1µM phenylephrine, and dilated by 1.9 ± 0.21% and 6.37 ± 0.64% in response to 1µM acetylcholine and flow rate increase from 0.5mL/min to 4mL/min, respectively (Figure 2). These results were significantly different from the response observed in control TEBV with EPC.
Figure 1. Functional characterization of iSMCs using calcium transience measurement. The graphs (right) show the change in calcium influx in the highlighted cells over time in response to the indicated substances as measured by the change in fluorescent intensity of the highlighted cells indicated in the image (left).
Figure 2. Functional vasoactivity of the TEBV was assessed by measuring the vessel diameter change in response to the addition of vasoactive drugs and increased flow rate. N=3. Error bars are the standard error mean (One * for p-value <0.05, Two * for p-value <0.01, Student’s t-test).
Conclusion: Human EPC transduced with MYOCD rapidly transdifferentiated into iSMC that showed significant resemblance to native SMCs in terms of its phenotype and genotype. The functional response of iSMC was demonstrated in 2D through calcium signaling activity, and in 3D through vasoactivity of the assembled iSMC-TEBV. Overall, iSMC produced in this study can be of a valuable cell source for TEBV production, which in turn can serve as an attractive vascular injury model for the study of vascular diseases.
References:
[1] Ingram, D. A. et al. Blood (2004), 104, 2752-2760
[2] Christoforou, N. et al. PLoS One (2013), 8, e63577.